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original library, was consequently screened at a much higher stringency to findthe most potent members. We identified a conserved family of sequences withextremely high membrane permeabilizing activity against lipid vesicles. Theseselected positives from the iteration library screen were synthesized and testedin antimicrobial and hemolytic assays. Despite their much higher vesicle per-meabilization activity, their antimicrobial activities are only slightly betterthan the positives identified in the original library. These results support theidea that antimicrobial activity is a complex bioactivity with important factorsthat extend beyond the simple concept of membrane permeabilization.

456-Pos Board B242Study of the Affinity of Structurally Different Antimicrobial Peptidesto Model Membranes and Their Ability to Induce Membrane Permeabili-zationKatia Regina Perez, Bruno Mattei, Tatiana M. Domingues,Antonio Miranda, Karin A. Riske.Universidade Federal de Sao Paulo, Sao Paulo, Brazil.Antimicrobial peptides are present in the immune system of flora and fauna,and have attracted attention due to their great potential of lytic action againstmembranes of a wide range of microorganisms. Here we study the mode of ac-tion of two structurally different synthetic cationic antimicrobial peptides: go-mesin, which adopts a b-hairpin structure due to two disulfide bridges, and thelinear peptide esculentin 1b(1-18), which acquires an a-helix structure uponbinding to amphiphilic surfaces. Additionally, different analogues of gomesinare also investigated: a linear one, and some other analogues in which specificresidues are replaced by alanine. Our focus is to understand the relationship be-tween peptide structure and its mode of action against liposomes composed ofdifferent molar ratios of a neutral and a negatively charged phospholipid,POPC/POPG, mimicking both bacterial and erythrocyte membranes. For thispurpose, several techniques were employed: fluorescence measurements ofthe leakage of carboxyfluorescein (CF) entrapped in vesicles, isothermal titra-tion calorimetry (ITC), and turbidity and zeta potential measurements. Thefluorescence studies showed that the ability of all peptides to promote CF leak-age from vesicles increases with the POPG molar ratio. The interaction of go-mesin and its analogues with POPC/POPGmembranes gives rise to exothermicpeaks, whose magnitude increases with the POPG molar ratio. Turbidity mea-surements showed that the binding of these antimicrobial peptides to the mem-brane surface is accompanied by vesicle aggregation. According to zetapotential measurements, vesicle aggregation seems to start when the membranesurface charge is neutralized by the peptide binding. Financial support: FA-PESP and INCT-FCx.

457-Pos Board B243Effect of Melittin and Gain-of-Function Melittin Analogs, Discovered byHigh-Throughput Screening, on Bilayer Properties: An Electrical Imped-ance Spectroscopy StudyGregory Wiedman1, William Wimley2, Peter Searson1, Kalina Hristova1.1Johns Hopkins University, Baltimore, MD, USA, 2Tulane University,New Orleans, LA, USA.Melittin, the main peptide component of European Honey Bee venom, is a 26-amino acid peptide that permeabilizes bacterial and mammalian cells, as wellas synthetic lipid vesicles. Melittin is one of the most studied pore forming pep-tides, and researchers hold it as a framework for designing engineered peptidespores. We have sought to optimize the potency of melittin, by applying orthog-onal high throughput screen strategies to select for gain-of-function analogs ofmelittin. Here we use electrochemical impedance spectroscopy (EIS) to com-pare the activity of melittin and one gain-of-function melittin analogue. WhileEIS studies of melittin show a decrease in admittance with a transient recoverywe find a strikingly different bilayer response to the melittin analogue- an ex-ponential decay with a half-life of 15-30 seconds. These experiments demon-strate the remarkable ability of the gain-of-function melittin analogue toform stable pores on lipid bilayers. Thus the engineered analogues havemany applications in biotechnology and as anti-cancer therapeutics.

458-Pos Board B244Relating Molecular-Level Events with Bacterial Killing by AntimicrobialPeptidesManuel N. Melo1,2, Rafael Ferre3, Carla Alves4, Miguel A.R.B. Castanho2.1University of Groningen, Groningen, Netherlands, 2Institute of MolecularMedicine, University of Lisbon, Portugal, 3University of Girona, Girona,Spain, 4Centro de Quı́mica da Madeira, University of Madeira, Portugal.We have collected several observations of threshold events in the interactionsof antimicrobial peptides (AMPs) with model membranes. These, however,

usually occur very close to full membrane coverage. Because such events arefrequently disruptive (lysis, pore formation, charge neutralization) we hypoth-esize a link between these and the antibacterial activity of the peptides. We thenestablish a mathematical relationship between the biophysical properties of

AMPs and threshold events in the bacte-rial membrane, and demonstrate thathigh membrane coverage is not onlyphysiologically plausible but also a likelyrequirement for AMP activity. In agree-ment, a co-occurrence of bacterial deathand surface charge neutralization was ob-served using zeta-potential measurementsof E. coli suspensions treated with theAMP BP100: Our model further allows the prediction of biologically-observed AMP activi-ties (the minimum inhibitory concentrations) from biophysical parameters ofpeptide-membrane interaction. This approach successfully predicted the activ-ities of two different AMPs against a set of susceptible strains.[1] M.N. Melo, R. Ferre and M.A.R.B. Castanho, Nature. Rev. Microbiol,2009, 7:245[2] C.S. Alves, M.N. Melo, et al., J. Biol. Chem., 2010, 285:27536.[3] R. Ferre*, M.N. Melo*, et al., Biophys. J., 2009, 96:1815.

459-Pos Board B245Role of the Side Chains in the Activity of Cyclic N-Substituted GlycineOligomersMarija Kosutic.Illinois Institute of Technology, Chicago, IL, USA.Non-natural mimics of antimicrobial peptides (AMPs) can be designed to dis-play chemical moieties analogous to the active side chains of natural peptides,while its abiotic backbone provides protection from proteolytic degradation. Adiscrete conformational structure of a molecule is commonly accepted to beanother prerequisite for potent antimicrobial activity. N-substituted glycineoligomers (peptoids) are among the most promising candidates as potentialanti-infectious agents due to their inherent ease of structural optimization.One of the approaches to rigidify the structure of peptoids is to introduce a co-valent limitation and make them cyclic. In the present work we investigate therole of side chains of three cyclic peptoids in their interactions with modelbacterial and cell membranes. The outer leaflets of Gram-positive and Gram-negative bacterial membranes were modeled with DPPG and Lipid A-Kdo2monolayers, respectively. The lipid monolayers at the air-liquid interfacewere studied using constant-pressure insertion assays, epifluorescence micros-copy (EFM), synchrotron X-ray reflectivity (XR) and grazing incident-anglediffraction (GID). We have found that both the electrostatic and hydrophobicforces play important roles in defining interactions of the peptoids with anioniclipid monolayers. Peptoids’ aryl side chains are responsible for the hydrophobicinteractions and serve as determinants for the differential activity of the pepti-domimetics. Our data suggest that bulkier groups promote an enhancedpeptoid insertion into the bacterial membrane monolayer mimics. This agreeswell with the higher antibacterial activity displayed by these compounds, indi-cating that bulkier groups, indeed, help peptoids in adopting rigid bioactiveconformation.

460-Pos Board B246A Plausible Molecular Mechanism for the Synergistic Activity ofTemporins at the Level of Lipopolysaccharide-Outer Membrane ofGram-Negative BacteriaMaria Luisa Mangoni1, Anirban Bhunia2, Surajit Bhattacharjya2.1University, Rome, Italy, 2Nanyang Technological University,Singapore, Singapore.Gene-encoded antimicrobial peptides (AMPs) represent attractive candidatesfor the development of a new generation of anti-infective agents.There is compelling evidence that unlike conventional antibiotics, most AMPsdo act by altering the membrane permeability of the target cell.However, before reaching it, they need to cross the microbial cell wall that,in Gram-negative bacteria, is surrounded by the lipopolysaccharide (LPS)-outer membrane, which forms an efficient barrier against a variety ofmolecules.In Nature, the frog skin temporins are among the shortest (10 to 14 residues)AMPs, with up to ten isoforms within the same specimen [1]. We have shownthat some of them (temporin-1Tb, TB, and temporin-1Tl, TL) have a synergisticeffect in killing Gram-negative bacteria [2]. This suggests an important strategyto overcome bacterial resistance due to the LPS layer. More precisely, we